NZ554183A

NZ554183A - Liposomal composition comprising an active ingredient for relaxing smooth muscle production and therapeutically use of said composition

Info

Publication number: NZ554183A
Application number: NZ554183A
Authority: NZ
Inventors: Andreas Wagner; Karola Vorauer-Uhl; Hermann Katinger
Original assignee: Polymun Scient Immunbio Forsch
Priority date: 2004-10-18
Filing date: 2005-10-14
Publication date: 2009-04-30
Also published as: KR20070069164A; DE502005008880D1; WO2006042701A1; AU2005296719A1; EA011391B1; PL1802277T3; JP5688199B2; ES2339577T3; EP1802277B1; US20090324698A1; EP1802277A1; CA2583332A1; PT1802277E; AU2005296719B2; CA2583332C; SI1802277T1; EA200700892A1; DK1802277T3; US8524274B2; KR101289917B1

Abstract

Disclosed is a pharmaceutical composition comprising an active ingredient included in liposomes, characterized in that the liposomes have, in their interior, an aqueous medium having a pH in the range of 2.5-5.5 and contain therein at least one protonated active ingredient which has a direct or indirect relaxing effect on the smooth muscles and an aqueous medium having a neutral or alkaline pH is present outside the liposomes, with the result that an H+ gradient is present between the inside and outside of the liposomes.

Description

554183 1 LIPOSOMAL COMPOSITION COMPRISING AN ACTIVE INGREDIENT FOR RELAXING SMOOTH MUSCLE, THE PRODUCTION OF THIS COMPOSITION AND THE THERAPEUTIC USE THEREOF FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions based on topically applicable active ingredients in liposomes, which enhance the blood flow 10 through tissues, in particular in the genital area, and to the production of such compositions and the use thereof.

BACKGROUND OF THE INVENTION Liposomes are known to be agents for the controlled release of pharmaceutical active ingredients (cf. for example overview by Ulrich, Biosci Rep. 20 02; 22(2):129-50 or WO 96/14083 for SOD in liposomes). The 20 formulation of local anaesthetics in topically applied liposomes is also known to the person skilled in the art; for example, US-4937078 describes liposomes which comprise customary sodium channel blockers, such as tetracaine, lidocaine, etc. Other known chemical 25 compounds are those which promote blood flow through tissues and have become known especially through their use for eliminating erectile dysfunction and impotence (cf. for example WO 94/28902 and EP 0967214 Al).

SUMMARY OF THE INVENTION While the known active ingredient preparations based on pyrazolopyrimidones and pyrazolopyrimidinones are 1 554183 2 present as formulations which can be administered either orally or intranasally, the object of the present invention is to provide a composition for effective topical use of such substances, preferably 5 directly in the genital area, in particular a formulation which permits an overall low but at the same time sufficiently high local dose of these active ingredients in the area of the female or male sex organs; and/or to provide the public with a useful 10 choice.

This object is achieved, according to the invention, by the provision of a liposomal system for topical, in particular transdermal and/or transmucosal, administration of active ingredients which relax the smooth muscles, especially those of the blood-supplying vessels in the sex organs. Such an effect can be triggered, for example, by an induced secretion of calcium ions.

DETAILED DESCRIPTION OF THE INVENTION In a first embodiment, the invention provides a pharmaceutical composition comprising an active 25 ingredient included in liposomes, characterized in that the liposomes have, in their interior, an aqueous medium having a pH in the range of 2.5-5.5 and contain therein at least one protonated active ingredient which has a direct or indirect relaxing effect on the smooth 30 muscles and an aqueous medium having a neutral or alkaline pH is present outside the liposomes, with the result that an H+ gradient is present between the inside and outside of the liposomes. intellectual property office of n.z. 1 8 MAK 2009 received 554183 2a Preferred formulations are those in which the active ingredient is preferably selected from the group 5 consisting of the prostaglandins, adenylate cyclases, cAMP, AMP, ATP, NO synthetases, nitric oxide (NO) , NO compounds, nitrates, guanylate cyclases, cGMP, GMP, GTP and phosphodiesterases, in particular sildenafil, tadalafil and vardenafil.

The liposomal formulation according to the invention achieves not only a temporary active ingredient depot 554183 3 in the surrounding tissue from which substance is continuously released but also better bioavailability and a longer half-life in comparison with systemic use.

The relaxing effect on the smooth muscle cells results in enhanced blood flow through the externally treated tissues, for example of the sex organs, and consequently in increased sensitivity and sensibility in sexual activities.

Active ingredients or active substances in the context of the present invention are in particular those substances which intervene in the cAMP or cGMP circulation and give rise to increased CA ion 15 secretion. These include, for example, substances such as papaverine and phentolamine which stimulate the cAMP pathway; nitric oxide (NO) which performs an important transmitter function and activates a guanylate cyclase which in turn forms cGMP; NO donors; nitroglycerin; 2 0 minoxidil; L-arginine; linsidomine (produced in the body by NO synthetase by conversion of arginine to citrulline}; molsidomine; phosphodiesterase inhibitors, such as, for example, sildenafil or sildenafil citrate, which intervenes in the cGMP pathway, a PDE5 receptor 25 being involved; prostaglandins, such as, for example, alprostadil (PGE-1), dinoprostone (PGE-2), which intervene in the cAMP circulation.

Where sildenafil is mentioned . below, tadalafil, 30 vardenafil and the acidic salts thereof, e.g. sildenafil citrate and vardenafil HC1-3H20 {vardenafil hydrochloride trihydrate), are also meant thereby, unless evident otherwise from the respective context. 554183 The scalable method disclosed in WO 02/36257 for active ingredient encapsulation has proved to be particular advantageous for the production and loading of the 5 liposomes with active ingredient, owing to its high efficiency in combination with extremely gentle conditions of the method. This method is typically used to produce unilamellar liposomes which have a lipid double-layer membrane and whose extremely good 10 skin penetrability has already been recognized and proven in earlier work. Other methods disclosed in the v prior art for the production and loading of liposomes can, however, also be used.

A maximum loading density can be achieved by active loading of the liposomes with active ingredients. This process can be divided into two main categories: loading of the membrane and loading of the intraliposomal aqueous phase. Active ingredients which 2 0 comprise protonatable groups, for example amino groups, can be included in liposomes by H+ gradient-controlled loading and then retained there in the protonated f state. For this type of active loading, the most important feature is the liposome membrane/liposome 25 medium partition coefficient. It was found that the octanol/buffer partition coefficient provides a good indication of the transmembrane diffusion of a substance and is therefore relevant for the loading with active ingredient or for the release profile.

On the basis of this theoretical model, liposomes having different lipid compositions, preferably comprising long-chain phospholipids and having low 554183 cholesterol concentrations, were produced in a suitable loading buffer, preferably in an ammonium sulphate or citric acid/sodium carbonate buffer. After the production of the liposomes in ammonium sulphate or 5 citrate buffer, the surrounding medium is modified, i.e. exchanged or diluted, optionally neutralized or made alkaline, and an H+ gradient is produced thereby between the intraliposomal buffer and the extraliposomal medium. After addition of an active 10 ingredient to the extraliposomal medium, the active ingredient migrates into the liposomes owing to this H+ gradient, is protonated there and remains stable in the liposomes.

On application of this technique, the extent of loading or the loading capacity is determined primarily by the ratio of H+ concentrations inside and outside the liposomes. In the experiments carried out, it was possible to achieve, with active ingredient/lipid 20 ratios in the range of 200-400 nmol of active ingredient per jimol of lipid, values similar to those known from the literature relating to actively loaded liposomes. An increase in the active ingredient concentration in the loading medium led to no increase 25 in the loading capacity.

The active loading described above is a three-stage process consisting of vesicle formation, addition of active ingredient and alka.linization. A further, object 30 of the invention was therefore to establish a one-stage production method which could be realized with the use of the crossflow module disclosed in WO 02/36257. For this purpose, the active ingredient was dissolved in ■ an 554183 6 H+-rich, aqueous phase, e.g. ammonium sulphate solution or citric acid solution, and included in liposomes by means of the crossflow injection technique and immediately thereafter the remaining external (= 5 extraliposomal), aqueous phase was diluted with a dilution buffer (e.g. 5% glucose solution in the ammonium sulphate system or citric acid/sodium carbonate pH 9.0-9.5 in the citrate system). It was found that the quality of the active ingredient-laden 10 liposomes can be improved simply by variation, in particular by reduction, of the cholesterol content in the vesicle membrane, especially with regard to the skin penetrability thereof, Where required or desired, the loading capacity can be further increased by raising the average liposome size from about 150-200 nm (as generally used in the experiments described herein) to 300-500 nm. In addition, the efficiency of the method, i.e. the amount 20 of liposomally included active ingredient per ml of suspension, can also be further increased by increasing the lipid concentration either during production or ( \ during the subsequent filtration of the vesicles.

If sildenafil is used as the active ingredient, the ammonium, sulphate/glucose solution system is preferable for active loading, since sildenafil is only poorly soluble or not soluble at all in citrate buffer. NH3, which is present in a reversible equilibrium with 30 ammonium sulphate in the intraliposomal aqueous medium, attempts to migrate out through the liposome membrane and to leave behind an H+. Sildenafil migrates in the opposite direction into the liposome, takes up the 554183 7 hydrogen ion H+, becomes more hydrophilic thereby and therefore remains in the. membrane. In this way, sildenafil can be efficiently loaded into liposomes. This applies in a similar manner also to the sildenafil 5 alternatives tadalafil and vardenafil.

For determining the best liposome formulation in relation to membrane flexibility and the associated skin penetration properties, various liposome 10 suspensions having different lipid compositions were prepared and tested. Phospholipids, optionally in combination with cholesterol, were primarily used. However, it is within the scope of the invention to replace or to supplement phospholipids with other 15 lipids, for example with glycolipids, cerebrosides, sulphatides or galactosides. Typical members of the lipids which can be used are, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelins, plasmalogens, glyceroglycolipids, ceramide, glycophingolipids and neutral glycophingolipids.

One possibility for improving the membrane fluidity 25 important for transdermal applications is to reduce the phase transition of the liposomal double layer membrane, which is determined chiefly by the length of the acyl chains of the phospholipids, the amount of cholesterol and the saturation of the. phospholipids. 30 For this reason, in one embodiment of the production method, DPPC, a phospholipid having an acyl chain length of 16 carbon atoms, was replaced by DMPC {chain length of 14 carbon atoms), which reduces the melting 554183 8 point TM from about 45°C to 31°C.

A second possibility for reducing the membrane rigidity and increasing the fluidity is to reduce the proportion 5 of cholesterol in the membrane. Starting from a DPPC : cholesterol ratio of 55:45 mol% {as described in the literature for liposome loading), the amount of cholesterol was gradually reduced to 38 or 30%, based on the total lipid content. A slight decrease in the 10 active ingredient loading was found in comparison with the results with higher cholesterol contents. However, these liposomes showed improved skin penetration properties and remained stable without significant active ingredient loss even in a long-term test over 15 weeks.

In contrast to discoveries in earlier work, it was also possible to produce stable cholesterol-free liposomes and successfully load them with active ingredient so 20 that, according to the invention, the cholesterol content is in a range of 0-50 mol% based on the total lipid content.

A third possibility for making liposomal membranes more 25 flexible is to replace the fully saturated DPPC or DMPC lipids with hen's egg phosphatidylcholine (E-PC), a natural lipid mixture with unsaturated phospholipids. In addition to stability problems with the use of these natural lipids, it was also necessary to produce the 30 liposomes under a nitrogen atmosphere. Nevertheless, these vesicles gave neither good results with respect to the vesicle size and homogeneity nor improvements in the skin penetration properties. 554183 It is within the scope of the present invention also to use alternative, functionally equivalent systems known to the person skilled in the art for the formation of 5 an H+ gradient. In this context, "functionally equivalent" is to be understood as meaning the ability to form an H+ gradient across the lipid double layer membrane of the liposomes and not to destroy the membrane integrity in the process, so that included, in 10 particular protonated, active ingredient remains stable - in the context of the stability criteria disclosed herein - in the liposomes.

For use of a liposomal sildenafil composition as a 15 therapeutic agent to be used topically, the liposomes are preferably mixed into a hydrogel, which is easier to apply to the skin than a pure suspension. However, it is within the scope of the present invention also to produce other galenical formulations for the sildenafil 20 liposomes and to apply them topically, in particular formulations in the form of solutions, lotions, emulsions, tinctures, sprays, ointments or creams. The person skilled in the art in this area is familiar with further possibilities as well as the required, 25 pharmaceutically admissible accompanying substances and additives for the production of the various galenical formulations.

In earlier experiments, for example, Carbopol 98 INF 30 (from Noveon), a hydrogel which can be used in very low concentrations, proved useful. It is admissible for pharmaceutical use, relatively cheap to acquire and available in large amounts. 554183 BRIEF DESCRIPTION OF THE FIGURE Fig. 1 shows a graph of the included amounts of sildenafil in DPPC/cholesterol liposomes as a 5 function of the vesicle size.

For better illustration, the invention is explained further with reference to the following examples.

DEFINITIONS The term ''comprising' as used in this specification and claims means ^consisting at least in part of, that is to say when interpreting independent claims including that term, the features prefaced by that term in each 15 claim all need to be present but other features can also be present.

Example 1: Production of sildenafil liposomes The liposomes are preferably produced by the known 20 crossflow method (WO 02/36257) using an aqueous phase suitable for the desired active ingredient, optionally at least a part of the active ingredient being initially taken in this aqueous phase and being included in the interior of the liposomes in the course 25 of the liposome formation. The subsequent dilution of the liposome suspension by means of neutral or alkaline dilution buffer, which preferably also contains active ingredient, produces an H+ gradient between the inside and outside of the liposomes, which both rapidly and 30 efficiently transports further protonatable active ingredient from the dilution buffer into the liposomes and retains the active ingredient already included in the course of the liposome formation. intellectual property office of n.z, 1 g MAK 2009 received 554183 10a Alternatively, it is also possible to choose a method in which, in a first step, buffer-filled liposomes without active ingredient are produced and the active ingredient is loaded into the liposomes actively via an H+ gradient, as described above, only after the liposome 1 intellectual property | office of n.z. 1 $ max 2009 received 554183 11 production. This procedure makes it possible to check the quality of the liposome suspension before loading with active ingredient.

Both techniques are very reproducible and permit the inclusion of any desired active ingredients in liposomes. They can also be carried out under extremely mild process conditions and make it possible completely to dispense with the use of possibly harmful 10 solvents and in particular with the use of shear forces for vesicle formation.

In addition, it is possible with this crossflow method to provide all reagents in sterile or germ-free form 15 and to carry out the liposome production and loading under aseptic conditions so that a sterile or germ-free product in the form of liposomes laden with active ingredient finally results.

Liposome production (according to WO 02/36257) in detail: The lipid mixture is dissolved in 96% ethanol and, depending on the choice of lipid or lipid composition, 25 at a temperature in the range of 25 to 60°C, for example at a temperature at 50 to 55 °C in the case of DPPC liposomes, with stirring. The buffer solutions, too, are preferably thermostatted at the same temperature, for example 55°C. While the polar, 30 aqueous phase (buffer) is pumped through the crossflow module by means of a pump, e.g. a peristaltic pump, the ethanol/lipid solution is simultaneously injected into the polar phase under a pressure which can be pre 554183 12 selected as desired.

Initial tests have shown that, depending on the active ingredient used, different buffer systems also exhibit 5 different suitability for the production of the H+ gradient which permits the active loading. Thus, for example with the use of sildenafil citrate as active ingredient, a buffer system of citric acid (intraliposomal) and an equimolar, neutral or slightly 10 basic buffer, such as, for example, citric acid/sodium carbonate pH 7.5-8.0 (extraliposomal), has proved to be disadvantageous since sildenafil citrate is insoluble or scarcely soluble in buffer systems of this type. On the other hand, sildenafil citrate dissolves very 15 readily in water, and it is for this reason that the active loading with ammonium sulphate gradients is preferred for this active ingredient.

By means of this method, liposomes are therefore 20 preferably formed in the presence of an ammonium sulphate buffer {preferably 125 mmol). After the vesicle formulation, the aqueous phase which has remained outside the liposomes, in this case the ammonium sulphate solution, is modified, for example 25 diluted by means of dilution buffer or replaced with a 5% glucose solution by means of diafiltration, with the result that small amphiphilic molecules, such as sildenafil, can be loaded into the liposomes and protonated there, while NH3 escapes from the liposomes 30 in the opposite direction. a) Two-stage variant: External loading of liposomes with sildenafil by means 554183 13 of H+ gradient. The best inclusion rates were achieved under the following conditions. The lipids (molar DPPC : cholesterol ratio = 55:45; in total 13 to 15 j^ntol per ml of aqueous phase) were dissolved in ethanol and this 5 solution was injected into 12 5 mM ammonium sulphate solution. After spontaneous vesicle formation, the remaining, external ammonium sulphate solution was replaced by a 5% glucose solution and sildenafil citrate was added. In this way, an H+ gradient formed 10 between the inside and outside of the lipid vesicles. pH values below 2.5 are less suitable owing to resultant hydrolysis . problems, and pH values greater than 5.5 are also not preferred, owing to the increasingly flat H+ gradient. An aqueous 125 mM 15 ammonium sulphate solution typically has a pH in the range of about 5-5.5.

After removal of sildenafil which has not been included by means of gel filtration, both the active ingredient 20 content and the lipid content were determined by means of rp-HPLC. Liposome size and distribution were determined by means of photon correlation spectroscopy (PCS).

Depending on the vesicle size, inclusion rates (represented as sildenafil/lipid ratio) of 160 to 230 nmol of sildenafil per (Jmol of total lipids (= DPPC + cholesterol) were achievable by this method of active loading. This converts to values of 1000-1500 (J.g of 30 active ingredient per ml of liposomal suspension.

In order to achieve an increase in the amount of 554183 14 liposomally included sildenafil, the sildenafil content in the glucose solution was increased. However, it was found that an excess of active ingredient could not improve the active ingredient/lipid ratio. The effective loading quantity in the case of active external loading via an H+ gradient therefore appears to be dependent primarily on the gradient and to a lesser extent on the initially taken active ingredient concentration. b) One-stage variant: The lipids (DPPC : cholesterol = 55:45 mol%) were dissolved in ethanol and the solution was injected into a sildenafil/ammonium sulphate solution (pH 3.5-4.5), 15 whereupon, immediately after spontaneous vesicle formation, a 5% glucose solution (pH 7), which comprised further sildenafil citrate, was added for dilution and alkalinization of the reaction mixture, i.e. of the resulting liposome suspension. As a result 2 0 of the production of this H+ gradient immediately after the vesicle formation, sildenafil is not only included in the liposomes in one step but is also retained there in a stable manner. The amount of sildenafil included liposomally in this manner was likewise in a range of 25 about 160 to 230 nmol of sildenafil per ^mol of total lipids (DPPC + cholesterol} - depending on the pH or H+ gradient.

The active ingredients tadalafil and vardenafil were 30 also loaded into liposomes in an analogous manner.

For comparison purposes, liposomally incorporated prostaglandin El was also produced under similar 554183 process conditions. Regarding the application effects, cf. Example 2.

Example 2: Use of liposomally incorporated active 5 ingredient A. Sildenafil, tadalafil, vardenafil in liposomes A formulation of 0.5 mg of the respective substance (calculated as salt-free active ingredient) in 10 liposomes per 1 ml of hydrogel Carbopol 981 NF was chosen as the pharmaceutical composition for use in a human experiment and used by test persons in an application amount of 0.5-1.5 ml per application, sildenafil and vardenafil being used in each case in 15 the form of their acidic salts (sildenafil citrate and vardenafil hydrochloride trihydrate, respectively).

The gel was used by male test persons for external application to the penis and by female test persons by 20 external vaginal and/or clitoral application.

Results: a) Male: Only shortly after application of the preparation, i.e. 25 within a few minutes, test persons experienced a pleasant, warm sensation and stronger sexual arousal. This was followed by a faster and stronger erection and substantially longer lasting stiffness of the penis in comparison with the effects usual or customary before, 30 without application of the preparation. Similar effects were achieved with the three active ingredient preparations. 554183 16 b) Female: Vaginal/clitoral application of the liposomal active ingredient gel immediately resulted in an effect which brought about increased blood flow, resulting in a 5 pleasant, warm sensation and subsequently increased production of vaginal secretion. In addition, the test persons reported slight contractions of the vaginal muscles (similar to those during orgasm), an enhanced sexual sensation during sexual intercourse and a 10 stronger sensation of orgasm.

The use of the liposomal active ingredient preparation not only results in sexual stimulation and enhancement of desire but also triggers stimulus more rapidly and 15 hence results in orgasm being reached more quickly.

Duration of action after single application: the effect begins immediately after application of the preparation, the sensations described above lasting for 2 0 up to 3.5 hours. All three preparations showed a clear effect; the test persons did not report any substantial, noticeable difference between the three different active ingredient preparations.

In contrast to the discoveries from Pfizer studies on women (cf. New York Times, 28 February 2004, "Pfizer Gives Up Testing Viagra on Women"') , at any rate the liposomal active ingredient preparations according to the invention appear to be clearly effective even in 30 women on external application. They can therefore also be used for the therapeutic treatment of female sexual dysfunction (FSD) , such as, for example, for the treatment of female sexual arousal disorder (FSAD). 554183 17 B. Prostaglandin El in liposomes Similar effects were described by the female test persons also after application of liposomal prostaglandin El. The applied dose was 0.1 mg-0.5 mg per 1 ml of gel and thus slightly below the dose of sildenafil, tadalafil and vardenafil. 554183 18

Claims

1. WHAT WE CLAIM IS: 10

2. Pharmaceutical composition comprising an active ingredient included in liposomes, characterized in that the liposomes have, in their interior, an aqueous medium having a pH in the range of 2.5-5.5 and contain therein at least one protonated active ingredient which has a direct or indirect relaxing effect on the smooth muscles and an aqueous medium having a neutral or alkaline pH is present outside the liposomes, with the result that an H+ gradient is present between the inside and outside of the liposomes. 15 20

3. Composition according to Claim 1, characterized in that an aqueous medium having a pH in the range of 5.0-5.5 is present inside the liposomes and an aqueous medium having a pH of 7 to 8 is present outside the liposomes.

4. Composition according to Claim 1 or 2, characterized in that the aqueous medium inside the liposomes is an aqueous buffer system. 25 4. Composition according to any one of Claims 1 to 3, characterized in that the aqueous medium inside the liposomes comprises an inorganic buffer. 30

5. Composition according to Claim 4, characterized in that the organic buffer is ammonium sulphate.

6. Composition according to any one of Claims 1 to 5, characterized in that the active ingredient is intellectual property office of n.z. 1 8 MAK 2009 received 554183 19 selected from the group consisting of the prostaglandins, adenylate cyclases, cAMP, AMP, ATP, NO synthetases, nitric oxide (NO), NO compounds, nitrates, guanylate cyclases, cGMP, 5 GMP, GTP and phosphodiesterases.

7. Composition according to any one of Claims 1 to 5, characterized in that the active ingredient is selected from the group consisting of the 10 substances sildenafil, tadalafil and vardenafil.

8. Composition according to any one of Claims 1 to 7, characterized in that it is applied locally. 15

9. Composition according to any one of Claims 1 to 8, characterized in that the medium outside the liposomes is a 5% glucose solution.

10. Composition according to any one of Claims 1 to 9, 20 characterized in that the liposomes are unilamellar and have a lipid double-layer membrane.

11. Composition according to any one of Claims 1 to 25 10, characterized in that the liposomes comprise phospholipids having an acyl chain length of at least 14 carbon atoms.

12. Composition according to Claim 11, characterized 30 in that the acyl chain length is at least 16 carbon atoms. 11ntf1 \ fctual property | office of n.z. 18 MAK 2009 received 554183 20 10

13. Composition according to any one of Claims 1 to 12, characterized in that the liposomes comprise a molar proportion of cholesterol of 0-50 %, based on the total amount of lipids.

14. Composition according to Claim 13, characterized in that the liposomes comprise a molar proportion of cholesterol of 30-45 %, based on the total amount of lipids.

15. Composition according to any one of Claims 1 to 14, characterized in that the liposomes have an average size in the range of 150 to 500 nm. 15

16. Composition according to any one of Claims 1 to 15, characterized in that the liposomes comprise the active ingredient in a concentration of at least 100 nmol per (Limol of lipid. 20

17. Composition according to Claim 16, characterized in that the liposomes comprise the active ingredient in a concentration of 150-400 nmol per jimol of lipid. 25

18. Composition according to any one of Claims 1 to 17, characterized in that it is present in the form of a suspension, lotion, emulsion, tincture or spray, gel, cream or ointment. 30

19. Composition according to Claim 18, characterized in that it is present in aseptic form. intellectual property office of n.z. 1 8 MAK 2009 received 554183 21

20. Method for the production of a pharmaceutical composition defined in any one of Claims 1 to 19, characterized in that liposomes having an aqueous medium in their interior are produced spontaneously by injection of an ethanolic lipid phase into an aqueous phase, whereupon the aqueous phase is modified, namely diluted, exchanged, neutralized or made alkaline, so that an H+ gradient forms between the inside and outside of the liposomes, and wherein an active ingredient a) is initially taken in the aqueous phase and is included in the liposomes in the course of the spontaneous liposome formation, and/or b) is added to the modified aqueous phase only after vesicle formation is complete and migrates along the H+ gradient into the liposomes.

21. Method according to Claim 20, characterized in that the modification of the aqueous phase is carried out immediately after liposome formation is complete.

22. Method according to Claim 20 or 21, characterized in that the aqueous phase which has a pH of 2.5-5.5 before the modification comprises an inorganic buffer and the modification is carried out by dilution of the aqueous phase with a neutral or alkaline buffer, with the result that a pH of 7-8 is achieved.

23. Method according to Claim 22, characterized in that the aqueous phase has a pH of 3.5-4.5 before intellectual property office of n.z. 1 8 mak 2009 554183 22 the modification and comprises ammonium sulphate and the modification is carried out by dilution of the aqueous phase with a 5 % glucose solution. 5

24. Method according to any one of Claims 20 to 23, characterized in that the lipid phase comprises phospholipids having an acyl chain length of at least 14 carbon atoms. 10

25. Method according to Claim 24, characterized in that the acyl chain length is at least 16 carbon atoms.

26. Method according to any one of Claims 20 to 25, 15 characterized in that the lipid phase comprises a molar proportion of cholesterol of 0-50 %, based on the total amount of lipids.

27. Method according to Claim 26, characterized in 20 that the lipid phase comprises a molar proportion of cholesterol of 30-45 %, based on the total amount of lipids.

28. Method according to any one of Claims 20 to 27, 25 characterized in that at least one substance having a relaxing effect on smooth muscles is initially taken as an active ingredient in the aqueous phase and/or is added to the modified aqueous phase after liposome formation is 30 complete.

29. Method according to Claim 28, characterized in that the substance is selected from the group \ intellectual property office of n.z. 1 8 MAK 2009 received 554183 23 10 consisting of the prostaglandins, adenylate cyclases, cAMP, AMP, ATP, NO synthetases, nitric oxide (NO), NO compounds, nitrates, guanylate cyclases, cGMP, GMP, GTP and phosphodiesterases.

30. Method according to Claim 28, characterized in that the substance is selected from the group consisting of the substances sildenafil, tadalafil and vardenafil.

31. Method according to any one of Claims 20 to 30, characterized in that the pharmaceutical composition is produced in the form of a suspension, lotion, emulsion, tincture or spray, 15 gel/' cream or ointment.

32. Method according to Claim 31, characterized in that the pharmaceutical formulation is produced in aseptic form. 20

33. Pharmaceutical composition according to any one of Claims 1 to 19, for use as a medicament for topical applications in the genital area. 25

34. Pharmaceutical composition according to any one of Claims 1 to 19, for use as a medicament for transdermal and/or transmucosal applications in the genital area. 30

35. Use of a pharmaceutical composition according to any one of Claims 1 to 19 for the production of a medicament for the prophylaxis and/or treatment of male erectile dysfunction. intellectual property office of n.z. 1 8 MAK 2009 received! 10 554183 24

36. Use of a pharmaceutical composition according to any one of Claims 1 to 19 for the production of a medicament for the treatment of female sexual dysfunction.

37. Use of a pharmaceutical composition according to any one of Claims 1 to 19 for the production of a medicament for the treatment of female sexual arousal disorder (FSAD).

38. Use of a pharmaceutical composition according to any one of Claims 1 to 19 for the production of a medicament for increasing sexual desire. 15

39. Use according to Claim 35 or 38 for the production of a medicament for external application to the penis.

40. Use according to any one of Claims 36 to 38 for 20 the production of a medicament for external vaginal and/or clitoral application.

41. A pharmaceutical composition as claimed in claim 1 substantially as herein described with reference to any example thereof and with or without 25 reference to the accompany drawings.

42. A method as claimed in claim 20 substantially as herein described with reference to any example thereof and with or without reference to the accompany drawings. 30

43. A use as claimed in any one of claims 35-38 substantially as herein described with reference to any example thereof and with or without reference to the accompany drawings. intellectual property OFFICE OF N.Z. 18 MAK 2009 >-»•— tvfr- r\